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Background: Nutrition administered as intermittent bolus feeds rather than continuously promotes greater protein synthesis rates in skeletal muscle and enhances lean growth in a neonatal piglet model. The molecular mechanisms responsible remain unclear., Objectives: We aimed to identify the insulin- and/or amino acid-signaling components involved in the enhanced stimulation of skeletal muscle by intermittent bolus compared to continuous feeding in neonatal pigs born at term., Methods: Term piglets (2-3 days old) were fed equal amounts of sow milk replacer [12.8 g protein and 155 kcal/(kg body weight · d)] by orogastric tube as intermittent bolus meals every 4 hours (INT) or by continuous infusion (CTS). After 21 days, gastrocnemius muscle samples were collected from CTS, INT-0 (before a meal), and INT-60 (60 minutes after a meal) groups (n = 6/group). Insulin- and amino acid-signaling components relevant to mechanistic target of rapamycin complex (mTORC) 1 activation and protein translation were measured., Results: Phosphorylation of the insulin receptor, IRS-1, PDK1, mTORC2, pan-Akt, Akt1, Akt2, and TSC2 was 106% to 273% higher in the skeletal muscle of INT-60 piglets than in INT-0 and CTS piglets (P  < 0.05), but phosphorylation of PTEN, PP2A, Akt3, ERK1/2, and AMPK did not differ among groups, nor did abundances of PHLPP, SHIP2, and Ubl4A. The association of GATOR2 with Sestrin1/2, but not CASTOR1, was 51% to 52% lower in INT-60 piglets than in INT-0 and CTS piglets (P  < 0.05), but the abundances of SLC7A5/LAT1, SLC38A2/SNAT2, SLC38A9, Lamtor1/2, and V-ATPase did not differ. Associations of mTOR with RagA, RagC, and Rheb and phosphorylation of S6K1 and 4EBP1, but not eIF2α and eEF2, were 101% to 176% higher in INT-60 piglets than in INT-0 and CTS piglets (P < 0.05)., Conclusions: The enhanced rates of muscle protein synthesis and growth with intermittent bolus compared to continuous feeding in a neonatal piglet model can be explained by enhanced activation of both the insulin- and amino acid-signaling pathways that regulate translation initiation., (© The Author(s) 2021. Published by Oxford University Press on behalf of the American Society for Nutrition.)

Extrauterine growth restriction in premature infants is largely attributed to reduced lean mass accretion and is associated with long-term morbidities. Previously, we demonstrated that prematurity blunts the feeding-induced stimulation of translation initiation signaling and protein synthesis in skeletal muscle of neonatal pigs. The objective of the current study was to determine whether the blunted feeding response is mediated by reduced responsiveness to insulin, amino acids, or both. Pigs delivered by cesarean section preterm (PT; 103 days, n = 25) or at term (T; 112 days, n = 26) were subject to euinsulinemic-euaminoacidemic-euglycemic (FAST), hyperinsulinemic-euaminoacidemic-euglycemic (INS), or euinsulinemic-hyperaminoacidemic-euglycemic (AA) clamps four days after delivery. Indices of mechanistic target of rapamycin complex 1 (mTORC1) signaling and fractional protein synthesis rates were measured after 2 h. Although longissimus dorsi (LD) muscle protein synthesis increased in response to both INS and AA, the increase was 28% lower in PT than in T. Upstream of mTORC1, Akt phosphorylation, an index of insulin signaling, was increased with INS but was 40% less in PT than in T. The abundances of mTOR·RagA and mTOR·RagC, indices of amino acid signaling, increased with AA but were 25% less in PT than in T. Downstream of mTORC1, eIF4E·eIF4G abundance was increased by both INS and AA but attenuated by prematurity. These results suggest that preterm birth blunts both insulin- and amino acid-induced activation of mTORC1 and protein synthesis in skeletal muscle, thereby limiting the anabolic response to feeding. This anabolic resistance likely contributes to the high prevalence of extrauterine growth restriction in prematurity. NEW & NOTEWORTHY Extrauterine growth faltering is a major complication of premature birth, but the underlying cause is poorly understood. Our results demonstrate that preterm birth blunts both the insulin-and amino acid-induced activation of mTORC1-dependent translation initiation and protein synthesis in skeletal muscle, thereby limiting the anabolic response to feeding. This anabolic resistance likely contributes to the reduced accretion of lean mass and extrauterine growth restriction of premature infants.

Background: Acute infection promotes skeletal muscle wasting and insulin resistance, but the effect of insulin on energy and substrate sensing in skeletal muscle of chronically infected neonates has not been studied., Methods: Eighteen 2-d-old pigs underwent cecal ligation and puncture (CLP) or sham surgery (CON) to induce a chronic infection for 5 d. On d 5, pancreatic-substrate clamps were performed to attain fasting or fed insulin levels but to maintain glucose and amino acids in the fasting range. Total fractional protein synthesis rates (K s ), translational control mechanisms, and energy sensing and degradation signal activation were measured in longissimus dorsi muscle., Results: In fasting conditions, CLP reduced K s and sirtuin 1 (SIRT1) and increased AMP-activated protein kinase α (AMPKα) activation and muscle RING-finger protein-1 (MuRF1). Insulin treatment increased K s and mitochondrial protein synthesis, enhanced translation activation, and reduced SIRT1 in CON. In contrast, in CLP, insulin treatment increased K s , protein kinase B (PKB) and Forkhead box O1 phosphorylation, antagonized AMPK activation, and decreased peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC-1α), MuRF1, and SIRT1., Conclusion: Energy and substrate sensing in skeletal muscle by the PKB-AMPK-SIRT1-PGC-1α axis is impacted by chronic infection in neonatal pigs and can be modulated by insulin.

Background: Sepsis induces loss of skeletal muscle mass by activating the ubiquitin proteasome (UPS) and autophagy systems. Although muscle protein synthesis in healthy neonatal piglets is responsive to amino acids (AA) stimulation, it is not known if AA can prevent the activation of muscle protein degradation induced by sepsis. We hypothesize that AA attenuate the sepsis-induced activation of UPS and autophagy in neonates., Methods: Newborn pigs were infused for 8 h with liposaccharide (LPS) (0 and 10 μg·kg(-1)·h(-1)), while circulating glucose and insulin were maintained at fasting levels; circulating AA were clamped at fasting or fed levels. Markers of protein degradation and AA transporters in longissimus dorsi (LD) were examined., Results: Fasting AA increased muscle microtubule-associated protein light 1 chain 3 II (LC3-II) abundance in LPS compared to control, while fed AA levels decreased LC3-II abundance in both LPS and controls. There was no effect of AA supplementation on activated protein kinase (AMP), forkhead box O1 and O4 phosphorylation, nor on sodium-coupled neutral AA transporter 2 and light chain AA transporter 1, muscle RING-finger protein-1 and muscle Atrophy F-Box/Atrogin-1 abundance., Conclusion: These findings suggest that supplementation of AA antagonize autophagy signal activation in skeletal muscle of neonates during endotoxemia., Competing Interests: Statement of financial support: The authors do not have any financial ties to products in the study or potential/perceived conflicts of interest to report.

The high efficiency of protein deposition during the neonatal period is driven by high rates of protein synthesis, which are maximally stimulated after feeding. In the current study, we examined the individual roles of amino acids and insulin in the regulation of protein synthesis in peripheral and visceral tissues of the neonate by performing pancreatic glucose-amino acid clamps in overnight-fasted 7-day-old pigs. We infused pigs (n = 8-12/group) with insulin at 0, 10, 22, and 110 ng kg(-0.66) min(-1) to achieve approximately 0, 2, 6 and 30 muU ml(-1) insulin so as to simulate below fasting, fasting, intermediate, and fed insulin levels, respectively. At each insulin dose, amino acids were maintained at the fasting or fed level. In conjunction with the highest insulin dose, amino acids were also allowed to fall below the fasting level. Tissue protein synthesis was measured using a flooding dose of L: -[4-(3)H] phenylalanine. Both insulin and amino acids increased fractional rates of protein synthesis in longissimus dorsi, gastrocnemius, masseter, and diaphragm muscles. Insulin, but not amino acids, increased protein synthesis in the skin. Amino acids, but not insulin, increased protein synthesis in the liver, pancreas, spleen, and lung and tended to increase protein synthesis in the jejunum and kidney. Neither insulin nor amino acids altered protein synthesis in the stomach. The results suggest that the stimulation of protein synthesis by feeding in most tissues of the neonate is regulated by the post-prandial rise in amino acids. However, the feeding-induced stimulation of protein synthesis in skeletal muscles is independently mediated by insulin as well as amino acids.

Sepsis has been associated with tumor necrosis factor alpha (TNF-alpha) and nitric oxide (NO) overproduction, insulin resistance, and a profound suppression of muscle protein synthesis. However, lesser suppression of muscle protein synthesis in neonatal pigs occurs in response to endotoxin (LPS) when glucose and amino acids are provided. We hypothesize that the LPS-induced TNF-alpha and NO overproduction down-regulates insulin signaling pathway activation in neonatal pigs in the presence of fed levels of insulin, glucose, and amino acids. In skeletal muscle, inducible NOS activity was increased in response to LPS infusion, but phosphorylation of the insulin receptor, insulin receptor substrate-1 (IRS-1), p42/p44 mitogen-activated protein kinase (MAPK), and protein kinase B, the association of IRS-1 with phosphatidylinositol 3-kinase (PI 3-kinase), and constitutive NOS activity were not altered. In liver, activation of the insulin receptor, IRS-1, and PI 3-kinase were not affected by LPS, but p42 MAPK phosphorylation was increased. The absence of a down-regulation in the insulin signaling cascade in muscle despite the LPS-induced increase in TNF-alpha and muscle iNOS, may contribute to the near-maintenance of muscle protein synthesis rates in the presence of glucose and amino acids in LPS-infused neonatal pigs.

Chronic treatment of growing pigs with porcine somatotropin (pST) promotes protein synthesis and doubles postprandial levels of insulin, a hormone that stimulates translation initiation. This study aimed to determine whether the pST-induced increase in skeletal muscle protein synthesis was mediated through an insulin-induced stimulation of translation initiation. After 7-10 days of pST (150 microg x kg(-1) x day(-1)) or control saline treatment, pancreatic glucose-amino acid clamps were performed in overnight-fasted pigs to reproduce 1) fasted (5 microU/ml), 2) fed control (25 microU/ml), and 3) fed pST-treated (50 microU/ml) insulin levels while glucose and amino acids were maintained at baseline fasting levels. Fractional protein synthesis rates and indexes of translation initiation were examined in skeletal muscle. Effectiveness of pST treatment was confirmed by reduced urea nitrogen and elevated insulin-like growth factor I levels in plasma. Skeletal muscle protein synthesis was independently increased by both insulin and pST. Insulin increased the phosphorylation of protein kinase B and the downstream effectors of the mammalian target of rapamycin, ribosomal protein S6 kinase, and eukaryotic initiation factor (eIF)4E-binding protein-1 (4E-BP1). Furthermore, insulin reduced inactive 4E-BP1.eIF4E complex association and increased active eIF4E.eIF4G complex formation, indicating enhanced eIF4F complex assembly. However, pST treatment did not alter translation initiation factor activation. We conclude that the pST-induced stimulation of skeletal muscle protein synthesis in growing pigs is independent of the insulin-associated activation of translation initiation.

Insulin and amino acids act independently to stimulate protein synthesis in skeletal muscle of neonatal pigs, and the responses decrease with development. The purpose of this study was to compare the separate effects of fed levels of INS and AA on the activation of signaling components leading to translation initiation and how these responses change with development. Overnight-fasted 6- (n = 4/group) and 26-day-old (n = 6/ group) pigs were studied during 1) euinsulinemic-euglycemiceuaminoacidemic conditions (controls), 2) euinsulinemic-euglycemichyperaminoacidemic clamps (AA), and 3) hyperinsulinemic-euglycemic-euaminoacidemic clamps (INS). INS, but not AA, increased the phosphorylation of protein kinase B (PKB) and tuberous sclerosis 2 (TSC2). Both INS and AA increased protein synthesis and the phosphorylation of mammalian target of rapamycin (mTOR), ribosomal protein S6 kinase-1, and eukaryotic initiation factor (eIF)4E-binding protein 1 (4E-BP1), and these responses were higher in 6-day-old compared with 26-day-old pigs. Both INS and AA decreased the binding of 4E-BP1 to eIF4E and increased eIF4E binding to eIF4G; these effects were greater in 6-day-old than in 26-day-old pigs. Neither INS nor AA altered the composition of mTORC1 (raptor, mTOR, and GbetaL) or mTORC2 (rictor, mTOR, and GbetaL) complexes. Furthermore, neither INS, AA, nor age had any effect on the abundance of Rheb and the phosphorylation of AMP-activated protein kinase and eukaryotic elongation factor 2. Our results suggest that the activation by insulin and amino acids of signaling components leading to translation initiation is developmentally regulated and parallels the developmental decline in protein synthesis in skeletal muscle of neonatal pigs.

Skeletal muscle protein synthesis is reduced in neonatal pigs in response to endotoxemia. To examine the role of insulin in this response, neonatal pigs were infused with endotoxin (LPS, 0 and 10 mug.kg(-1).h(-1)), whereas glucose and amino acids were maintained at fasting levels and insulin was clamped at fasting or fed (2 or 10 muU/ml) levels. Fractional rates of protein synthesis and translational control mechanisms were examined in longissimus dorsi muscle and liver. In the presence of fasting insulin, LPS reduced muscle protein synthesis (-29%), and increasing insulin to fed levels accelerated muscle protein synthesis in both groups (controls, +44%; LPS, +64%). LPS, but not insulin, increased liver protein synthesis by +28%. In muscle of fasting neonatal pigs, LPS reduced 4E-BP1 phosphorylation and eIF4E to eIF4G binding. In muscle of controls, but not LPS pigs, raising insulin to fed levels increased 4E-BP1 and S6K1 phosphorylation and eIF4E to eIF4G binding. In muscle and liver, neither LPS nor insulin altered eIF2B activity. eEF2 phosphorylation decreased in response to insulin in both LPS and control animals. The results suggest that, in endotoxemic neonatal animals, the response of protein synthesis to insulin is maintained despite suppression of mTOR-dependent translation initiation and eIF4E availability for eIF4F assembly. Maintenance of an anabolic response to the feeding-induced rise in insulin likely exerts a protective effect for the neonate to the catabolic processes induced by sepsis.

The rapid growth of neonates is driven by high rates of skeletal muscle protein synthesis. This high rate of protein synthesis, which is induced by feeding, declines with development. Overnight-fasted 7- and 26-day-old pigs either remained fasted or were refed, and the abundance and phosphorylation of growth factor- and nutrient-induced signaling components that regulate mRNA translation initiation were measured in skeletal muscle and liver. In muscle, but not liver, the activation of inhibitors of protein synthesis, phosphatase and tensin homolog deleted on chromosome 10, protein phosphatase 2A, and tuberous sclerosis complex 1/2 increased with age. Serine/threonine phosphorylation of the insulin receptor and insulin receptor substrate-1, which downregulates insulin signaling, and the activation of AMP-activated protein kinase, an inhibitor of protein synthesis, were unaffected by age and feeding in muscle and liver. Activation of positive regulators of protein synthesis, mammalian target of rapamycin (mTOR), ribosomal protein S6 kinase 1 (S6K1), and eIF4E-binding protein-1 (4E-BP1) decreased with age in muscle but not liver. Feeding enhanced mTOR, S6K1, and 4E-BP1 activation in muscle, and this response decreased with age. In liver, activation of S6K1 and 4E-BP1, but not mTOR, was increased by feeding but was unaffected by age. Raptor abundance and the association between raptor and mTOR were greater in 7- than in 26-day-old pigs. The results suggest that the developmental decline in skeletal muscle protein synthesis is due in part to developmental regulation of the activation of growth factor and nutrient-signaling components.

Sepsis promotes insulin resistance and reduces protein synthesis in skeletal muscle of adults. The effect of sepsis on insulin-stimulated muscle protein synthesis has not been determined in neonates, a highly anabolic population that is uniquely sensitive to insulin. Overnight fasted neonatal pigs were infused for 8 h with endotoxin [lipopolysaccharide (LPS), 0 and 10 mug.kg(-1).h(-1)]. Glucose and amino acids were maintained at fasting levels, insulin was clamped at either fasting or fed (2 or 10 muU/ml) levels, and fractional protein synthesis rates were determined at the end of the infusion. LPS infusion induced a septic-like state, as indicated by a sustained elevation in body temperature, heart rate, and cortisol. At fasting insulin levels, LPS reduced fractional protein synthesis rates in gastrocnemius muscle (-26%) but had no effect on the masseter and heart. By contrast, LPS stimulated liver protein synthesis (+28%). Increasing insulin to fed levels accelerated protein synthesis rates in gastrocnemius (controls by +38%, LPS by +60%), masseter (controls by +50%, LPS by +43%), heart (controls by +34%, LPS by +40%), and diaphragm (controls by +54%, LPS by +29%), and the response to insulin was similar in LPS and controls. Insulin did not alter protein synthesis in liver, kidney, or jejunum in either group. These findings suggest that acute endotoxemia lowers basal fasting muscle protein synthesis in neonates but does not alter the response of protein synthesis to insulin.

The high efficiency of protein deposition during the neonatal period is driven by high rates of protein synthesis, which are maximally stimulated after feeding. Infusion of amino acids, but not insulin, reproduces the feeding-induced stimulation of liver protein synthesis. To determine whether amino acid-stimulated liver protein synthesis is independent of insulin in neonates, and to examine the role of amino acids and insulin in the regulation of translation initiation in neonatal liver, we performed pancreatic glucose-amino acid clamps in overnight-fasted 7-day-old pigs. Pigs (n = 9-12/group) were infused with insulin at 0, 10, 22, and 110 ng.kg(-0.66).min(-1) to achieve 0, 2, 6, and 30 microU/ml insulin, respectively. At each insulin dose, amino acids were maintained at fasting or fed levels or, in conjunction with the highest insulin dose, allowed to fall to below fasting levels. Insulin had no effect on the fractional rate of protein synthesis in liver. Amino acids increased fractional protein synthesis rates in liver at each dose of insulin, including the 0 microU/ml dose. There was a dose-response effect of amino acids on liver protein synthesis. Amino acids and insulin increased protein S6 kinase and 4E-binding protein 1 (4E-BP1) phosphorylation; however, only amino acids decreased formation of the inactive 4E-BPI.eukaryotic initiation factor-4E (eIF4E) complex. The results suggest that amino acids regulate liver protein synthesis in the neonate by modulating the availability of eIF4E for 48S ribosomal complex formation and that this response does not require insulin.

Feeding stimulates protein synthesis in skeletal muscle and liver of neonates and this response can be reproduced in muscle by the infusion of insulin or amino acids and in liver by the infusion of amino acids, but not insulin. Activation of insulin signaling components leading to translation initiation is associated with the feeding-induced stimulation of muscle protein synthesis in neonates. In this study, we examined the individual roles of insulin and amino acids in the activation of insulin signaling components leading to translation initiation, specifically, the insulin receptor (IR), insulin receptor substrate 1 (IRS-1), phosphatidylinositol 3-kinase (PI 3-kinase), protein kinase B (PKB) and ribosomal protein S6. Insulin secretion was blocked by somatostatin in food-deprived, 7-d-old pigs (n=8-12/group); insulin was infused to achieve plasma levels of approximately 0, 17, 52, and 255 pmol/L (approximately 0, 2, 6, 30 microU/mL), and amino acids were clamped at food-deprived or fed levels. In skeletal muscle, insulin increased the activation of IR, IRS-1, PI 3-kinase, PKB and S6 and stimulated protein synthesis. In liver, insulin increased the activation of IR, IRS-1, PI 3-kinase, PKB and S6, but had no effect on protein synthesis. Raising amino acids from the food-deprived to the fed level did not alter the insulin-induced activation of IR, IRS-1, PI 3-kinase and PKB but increased S6 phosphorylation and protein synthesis in skeletal muscle and liver. The results suggest that the stimulation of protein synthesis in muscle by insulin involves activation of insulin signaling components, and the stimulation of protein synthesis in muscle and liver by amino acids occurs by mechanisms independent of the early steps of this pathway. Furthermore, amino acids do not alter the insulin-stimulated activation of early steps in the insulin signaling pathway.

Previous studies have shown that intravenous infusion of insulin and/or amino acids reproduces the feeding-induced stimulation of muscle protein synthesis in neonates and that insulin and amino acids act independently to produce this effect. The goal of the present study was to delineate the regulatory roles of insulin and amino acids on muscle protein synthesis in neonates by examining translational control mechanisms, specifically the eukaryotic translation initiation factors (eIFs), which enable coupling of initiator methionyl-tRNAi and mRNA to the 40S ribosomal subunit. Insulin secretion was blocked by somatostatin in fasted 7-day-old pigs (n = 8-12/group), insulin was infused to achieve plasma levels of approximately 0, 2, 6, and 30 microU/ml, and amino acids were clamped at fasting or fed levels or, at the high insulin dose, below fasting. Both insulin and amino acids increased the phosphorylation of ribosomal protein S6 kinase (S6K1) and the eIF4E-binding protein (4E-BP1), decreased the binding of 4E-BP1 to eIF4E, increased eIF4E binding to eIF4G, and increased fractional protein synthesis rates but did not affect eIF2B activity. In the absence of insulin, amino acids had no effect on these translation initiation factors but increased the protein synthesis rates. Raising insulin from below fasting to fasting levels generally did not alter translation initiation factor activity but raised protein synthesis rates. The phosphorylation of S6K1 and 4E-BP1 and the amount of 4E-BP1 bound to eIF4E and eIF4E bound to eIF4G were correlated with insulin level, amino acid level, and protein synthesis rate. Thus insulin and amino acids regulate muscle protein synthesis in skeletal muscle of neonates by modulating the availability of eIF4E for 48S ribosomal complex assembly, although other processes also must be involved.

Infusion of physiological levels of insulin and/or amino acids reproduces the feeding-induced stimulation of muscle protein synthesis in neonates. To determine whether insulin and amino acids independently stimulate skeletal muscle protein synthesis in neonates, insulin secretion was blocked with somatostatin in fasted 7-day-old pigs (n = 8-12/group) while glucose and glucagon were maintained at fasting levels and insulin was infused to simulate either less than fasting, fasting, intermediate, or fed insulin levels. At each dose of insulin, amino acids were clamped at either the fasting or fed level; at the highest insulin dose, amino acids were also reduced to less than fasting levels. Skeletal muscle protein synthesis was measured using a flooding dose of l-[4-(3)H]phenylalanine. Hyperinsulinemia increased protein synthesis in skeletal muscle during hypoaminoacidemia and euaminoacidemia. Hyperaminoacidemia increased muscle protein synthesis during hypoinsulinemia and euinsulinemia. There was a dose-response effect of both insulin and amino acids on muscle protein synthesis. At each insulin dose, hyperaminoacidemia increased muscle protein synthesis. The effects of insulin and amino acids on muscle protein synthesis were largely additive until maximal rates of protein synthesis were achieved. Amino acids enhanced basal protein synthesis rates but did not enhance the sensitivity or responsiveness of muscle protein synthesis to insulin. The results suggest that insulin and amino acids independently stimulate protein synthesis in skeletal muscle of the neonate.

In neonatal pigs, the feeding-induced stimulation of protein synthesis in skeletal muscle, but not liver, can be reproduced by insulin infusion when essential amino acids and glucose are maintained at fasting levels. In the present study, 7- and 26-day-old pigs were studied during 1) fasting, 2) hyperinsulinemic-euglycemic-euaminoacidemic clamps, 3) euinsulinemic-euglycemic-hyperaminoacidemic clamps, and 4) hyperinsulinemic-euglycemic-hyperaminoacidemic clamps. Amino acids were clamped using a new amino acid mixture enriched in nonessential amino acids. Tissue protein synthesis was measured using a flooding dose of L-[4-(3)H]phenylalanine. In 7-day-old pigs, insulin infusion alone increased protein synthesis in various skeletal muscles (from +35 to +64%), with equivalent contribution of myofibrillar and sarcoplasmic proteins, as well as cardiac muscle (+50%), skin (+34%), and spleen (+26%). Amino acid infusion alone increased protein synthesis in skeletal muscles (from +28 to +50%), also with equivalent contribution of myofibrillar and sarcoplasmic proteins, as well as liver (+27%), pancreas (+28%), and kidney (+10%). An elevation of both insulin and amino acids did not have an additive effect. Similar qualitative results were obtained in 26-day-old pigs, but the magnitude of the stimulation of protein synthesis by insulin and/or amino acids was lower. The results suggest that, in the neonate, the stimulation of protein synthesis by feeding is mediated by either amino acids or insulin in most tissues; however, the feeding-induced stimulation of protein synthesis in skeletal muscle is uniquely regulated by both insulin and amino acids.

In neonatal animals, feeding stimulates skeletal muscle protein synthesis, a response that declines with development. Both the magnitude of the feeding response and its developmental decline can be reproduced by insulin infusion, suggesting that an altered responsiveness to insulin is a primary determinant of the developmental decline in the stimulation of protein synthesis by feeding. In this study, 7- and 26-day-old pigs were either fasted overnight or fed porcine milk after an overnight fast. We examined the abundance and degree of tyrosine phosphorylation of the insulin receptor (IR), insulin receptor substrate-1 (IRS-1), and IRS-2 in skeletal muscle and, for comparison, liver. We also evaluated the association of IRS-1 and IRS-2 with phosphatidylinositol 3-kinase (PI 3-kinase). The abundance of IR protein in muscle was twofold higher at 7 than at 26 days, but IRS-1 and IRS-2 abundances were similar in muscle of 7- and 26-day-old pigs. The feeding-induced phosphorylations were greater at 7 than at 26 days of age for IR (28- vs. 13-fold), IRS-1 (14- vs. 8-fold), and IRS-2 (21- vs. 12-fold) in muscle. The associations of IRS-1 and IRS-2 with PI 3-kinase were also increased by refeeding to a greater extent at 7 than at 26 days (9- vs. 5-fold and 6- vs. 4-fold, respectively). In liver, the abundance of IR, IRS-1, and IRS-2 was similar at 7 and 26 days of age. Feeding increased the activation of IR, IRS-1, IRS-2, and PI 3-kinase in liver only twofold, and these responses were unaffected by age. Thus our findings demonstrate that the feeding-induced activation of IR, IRS-1, IRS-2, and PI 3-kinase in skeletal muscle decreases with development. Further study is needed to ascertain whether the developmental decline in the feeding-induced activation of early insulin-signaling components contributes to the developmental decline in translation initiation in skeletal muscle.

We recently demonstrated in neonatal pigs that, with amino acids and glucose maintained at fasting levels, the stimulation of protein synthesis in longissimus dorsi muscle with feeding can be reproduced by a physiological rise in insulin alone. In the current report, we determine whether the response of protein synthesis to insulin in the neonatal pig is 1) present in muscles of different fiber types, 2) proportional in myofibrillar and sarcoplasmic proteins, 3) associated with increased translational efficiency and ribosome number, and 4) present in other peripheral tissues and in viscera. Hyperinsulinemic-euglycemic-amino acid clamps were performed in 7- and 26-day-old pigs infused with 0, 30, 100, or 1,000 ng. kg(-0.66). min(-1) of insulin to reproduce insulin levels present in fasted, fed, refed, and supraphysiological conditions, respectively. Tissue protein synthesis was measured using a flooding dose of L-[4-(3)H]phenylalanine. Insulin increased protein synthesis in gastrocnemius muscle and, to a lesser degree, masseter muscle. The degree of stimulation of protein synthesis by insulin was similar in myofibrillar and sarcoplasmic proteins. Insulin increased translational efficiency but had no effect on ribosome number in muscle. All of these insulin-induced changes in muscle protein synthesis decreased with age. Insulin also stimulated protein synthesis in cardiac muscle and skin but not in liver, intestine, spleen, pancreas, or kidney. The results support the hypothesis that insulin mediates the feeding-induced stimulation of myofibrillar and sarcoplasmic protein synthesis in muscles of different fiber types in the neonate by increasing the efficiency of translation. However, insulin does not appear to be involved in the feeding-induced stimulation of protein synthesis in visceral tissues. Thus different mechanisms regulate the growth of peripheral and visceral tissues in the neonate.

Somatotropin (ST) administration enhances protein deposition and elicits profound metabolic responses, including hyperinsulinemia. To determine whether the anabolic effect of ST is due to hyperinsulinemia, pair-fed weight-matched growing swine were treated with porcine ST (150 microg x kg body wt(-1) x day(-1)) or diluent for 7 days (n = 6/group, approximately 20 kg). Then pancreatic glucose-amino acid clamps were performed after an overnight fast. The objective was to reproduce the insulin levels of 1) fasted control and ST pigs (basal insulin, 5 microU/ml), 2) fed control pigs (low insulin, 20 microU/ml), and 3) fed ST pigs (high insulin, 50 microU/ml). Amino acid and glucose disposal rates were determined from the infusion rates necessary to maintain preclamp blood levels of these substrates. Whole body nonoxidative leucine disposal (NOLD), leucine appearance (R(a)), and leucine oxidation were determined with primed, continuous infusions of [(13)C]leucine and [(14)C]bicarbonate. ST treatment was associated with higher NOLD and protein balance and lower leucine oxidation and amino acid and glucose disposals. Insulin lowered R(a) and increased leucine oxidation, protein balance, and amino acid and glucose disposals. These effects of insulin were suppressed by ST treatment; however, the protein balance remained higher in ST pigs. The results show that ST treatment inhibits insulin's effects on protein metabolism and indicate that the stimulation of protein deposition by ST treatment is not mediated by insulin. Comparison of the protein metabolic responses to ST treatment during the basal fasting period with those in the fully fed state from a previous study suggests that the mechanism by which ST treatment enhances protein deposition is influenced by feeding status.

The elevated rate of muscle protein deposition in the neonate is largely due to an enhanced stimulation of skeletal muscle protein synthesis by feeding. To examine the role of insulin in this response, hyperinsulinemic-euglycemic-amino acid clamps were performed in 7- and 26-day-old pigs. Pigs were infused with 0, 30, 100, or 1,000 ng . kg-0.66 . min-1 of insulin to mimic the plasma insulin levels observed under fasted, fed, refed, and supraphysiological conditions, respectively. Whole body amino acid disposal was determined from the rate of infusion of an amino acid mixture necessary to maintain plasma essential amino acid concentrations near their basal fasting levels. A flooding dose of L-[4-3H]phenylalanine was used to measure skeletal muscle protein synthesis. Whole body amino acid disposal increased progressively as the insulin infusion rate increased, and this response was greater in 7- than in 26-day-old pigs. Skeletal muscle protein synthesis was stimulated by insulin, and this response was maximal at a low insulin infusion rate (30 ng . kg-0.66 . min-1). The stimulation of muscle protein synthesis by insulin was also greater in 7- than in 26- day-old pigs. These data suggest that muscle protein synthesis is more sensitive to insulin than whole body amino acid disposal. The results further suggest that insulin is a central regulatory factor in the elevated rate of muscle protein deposition and the increased response of skeletal muscle protein synthesis to feeding in the neonate.

Neonatal animals utilize their dietary amino acids for protein accretion with high efficiency, and this efficiency declines during early life. The factors responsible for this developmental change are unknown. Our objectives were to determine whether amino acid (AA) utilization is stimulated by insulin in the neonate and whether this response changes during the suckling period. Two hyperinsulinemic-euglycemic clamp infusion studies, using 10-2,000 ng insulin.kg-0.66.min-1, were performed in 7- and 26-day-old pigs. In study I, no AA were provided during the infusion, and the resultant decline in plasma AA levels was defined. In study II, plasma AA were clamped at near-fasting levels, and whole body utilization of exogenous AA was determined by measuring the rate of infusion of an AA mixture necessary to maintain basal plasma lysine concentrations. In study I, the half-maximal effective dose (ED50) for the fall in AA concentrations with increasing plasma insulin concentration was lower in 7- than in 26-day-old pigs, and the nadir in AA concentration was achieved by only 20 microU/ml insulin. In study II, the utilization of exogenous AA during hyperinsulinemic-euglycemic AA clamps exhibited a higher maximum response (Rmax) (49 vs. 26 mumol AAtotal.min-1.kg-1) and a lower ED50 (18 vs. 45 microU insulin/ml) in 7- than in 26-day-old pigs. Plasma urea nitrogen concentrations did not rise with increasing insulin and AA infusion rates. These results indicate that insulin stimulates the utilization of exogenous AA in neonatal pigs and that both the insulin sensitivity and responsiveness of AA utilization decline over the suckling period. The infused AA were likely utilized for protein accretion.

Plasma amino acid concentrations fall during insulin infusion. Amino acid concentrations can be maintained using an infusion of amino acids if their plasma concentration can be determined within a few minutes. We developed a spectrophometric assay which determines the total concentration of all three branched-chain amino acids in plasma within 1 min. The enzyme leucine dehydrogenase oxidatively deaminates leucine, isoleucine, and valine, with stoichiometric reduction of NAD that is measured using a spectrophotometer. The assay was developed in both a kinetic and end-point format. For the kinetic assay the buffer conditions were formulated to obtain equivalent rates with all three amino acids so that it could be used in samples containing unknown mixtures. For the end-point assay additional enzyme was added so that an end-point could be reached within 1 min. The application of the kinetic assay for "clamping" the branched-chain amino acids during hyperinsulinemic euglycemic clamps in humans is demonstrated.

To compare the sensitivity of muscle protein synthesis to food intake in neonatal and weaned rats, 5- and 16-day-old suckled rats and 28-day-old weaned rats were either fed, fasted for 8-10 h, or refed for 1-4 h after an 8-h fast. Protein synthesis was measured in vivo in soleus and plantaris muscles with a large dose of L-[4-3H]phenylalanine. In fed rats, fractional rates of protein synthesis (KS) decreased with age. Fasting decreased KS, and refeeding increased KS most in 5-day-old animals, less in 16-day-old rats, and least in 28-day-old rats. In 5-day-old rats, there were no differences in KS between soleus and plantaris muscles in the fed state and after fasting and refeeding; at 28 days, KS was higher in soleus than in plantaris in fed rats, and the soleus did not respond to fasting and refeeding. In rats at all three ages, the concentration of most plasma amino acids decreased during fasting; when 5-day-old rats were refed, plasma amino acid concentrations increased, but not to the levels in the fed state. Plasma insulin concentrations increased with age. Plasma insulin concentrations decreased more rapidly with fasting and increased more extensively with refeeding in 5-day-old rats than in older rats. These results suggest that muscle protein synthesis is more responsive to food intake in young suckled rats than in older suckled or weaned rats; this increased responsiveness is accompanied by greater changes in circulating insulin concentrations.